Drugs of abuse, such as cocaine, potentiate neuroinflammation associated with HIV-1 infection despite the advent of combined anti-retroviral therapy (cART). One of the essential reasons is that cART has minimal effect on the expression of viral gene products such as Tat or gp120 that continue to be present in tissues such as the brain. Neuroinflammation involving robust microglial activation underlies HIV-associated neurocognitive disorders (HAND). Therefore strategies aimed at blocking inflammation could be considered as possible treatment options for ameliorating pathogenesis of HAND in the context of cocaine abuse. Emerging evidence points to the role of microRNAs (miRs) as regulators of glial activation (inflammation). Preliminary in vitro and in vivo findings from our lab have demonstrated that: a) HIV Tat mediated activation of microglia involves downregulation of miR-223 and, b) cocaine-mediated activation of these cells involves downregulation of miR-124. Reciprocally, overexpression of miRs-223 & -124 was found to rescue Tat and cocaine-mediated activation of microglia, respectively. We thus hypothesized that miRs-223 and -124 could thus be developed as therapeutic agents for ameliorating HIV Tat and cocaine-mediated neuroinflammation. Finding an effective and a less invasive method for drug delivery into the brain however, remains one of the major challenges in the field. The goal of the current proposal is to design and develop miR(s)-loaded extracellular vesicles (EV) for in vivo delivery as a means to ameliorate HIV protein (Tat) & cocaine-induced microglia activation in the brain. This proposal is focused specifically on the generation and optimization of engineered EVs containing miRs (-223 & -124) and CNS targeted peptides (to facilitate brain localization) with the ultimate goal of testing their efficacy in blocking microglial activation mediated by HIV Tat and cocaine, in a rodent model of HAND. EVs are becoming well recognized as cell-cell communication conduits that deliver the cargo containing miR, mRNA and proteins to the neighboring/distant cells. Our preliminary studies have demonstrated that HIV Tat and cocaine mediated downregulation of miRs-223 & -124 resulted in microglia activation via upregulation of NLRP3 (a target of miR-223) and KLF4 & TLR4 (targets of miR-124). Based on these findings we hypothesize that intranasal delivery of EVs (from dicer knock out dendritic cells) that are loaded with miRs (-223 & -124) and CNS target peptides, have the potential to abrogate Tat and cocaine induced-microglial activation. This hypothesis will be tested via two specific aims. Specific Aim 1: To design, engineer and optimize miR-depleted EVs loaded with miRs-223 and -124 in vitro for their ability to block HIV Tat and cocaine-mediated microglial activation. Specific Aim 2: To assess in vivo the role of brain-targeting miR-depleted-EVs loaded with miRs-223 and -124 in ameliorating HIV Tat & cocaine -mediated microglial activation. Our long-term goal is to develop brain-targeting miR-depleted-EV-based RNA drug delivery in vivo as a therapeutic for use in future preclinical trials for the treatment of HAND in cocaine abusers.